USPTO Patents Application 09636530

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WORLD INTELLECTUAL PROPERTY ORGANIZATIOM
International Bureau

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(5!) International Patent Classification ^ :

(t I) International Publication Number:

WO 93/06845

A61K 37/02, 37/36, G07K 7/10

Al

(43) International Publication Date:

15 April 1993(I5;04.93)

(21) rntemational Application Number: PCT''US92 '08477

(22) International niing Date: 9 October 1992 (09. 10.92)

(30)Prioritv data:

773,098

10 October 1991 (10.10.91) US

(71X72) Applicant and Inventor: PANG. Peter. K.. T, (US/CA):
52225 Range Road 232. 205 Carriage Lane, Sherwood
. Park, Alberta T8A 2A6 (CA).

(72) Inventor; and

(75) Ihventor/Applicint ifor US onlvj : SHAN. Jie [CN^CA]: ,
10615-83 Avenue, #105. Edmonton. Alberta T6E 2E3
(CA).

(74) Agent: MURRAY. Robert, B.; Nikaido. Mannelstein,
Murray & Oram, Metropolitan Square. fB^e 330, G
Street 'Lobby, 655 I5th Street, NAV.. Washington, DC
•20005-5701 (US).

(81) Designated States: AT, AU, BB. BG, BR, CA, GH. CS,
DE. DK, ES, FI, GB, HU. JP, KP, KR, LK, LU, MG,
MN. MW. ML. NO, PL, RO, RU, SD, SE, US, Euro-
pean patent (AT, BE, CH, DE, DK, ES, FR, GB, GR,
IE, IT. LU. MC. NL, SE). OAPI patent (BF, BJ, CP,
CG. CI, CM, GA, GN. ML, MR, SN, TD, TG).

Published

IVifh tfuernanonal search report.

(54) Title: PARATHYROID HORMONE ANALOGUES SUBSTITUTED AT aa^5.^6.^" AND USE IN OSTEOPOROSIS
TREATMENT

(57) Abstract - ' ' ^

Analogues of bovine and human parathyroid hormone, wherein nventy-fifth, twenty-sixth and twenty-seventh" pbtitions of :
the natural hormone. Arg-Lys-Lys- each have been substituted with Ala. Asn, Asp, Cys, Gin, Glu, Gly. His, lie. Leu, Met, Phe,
Pro. Sen Thr. Trp, Tyr or Yal have been found to retain bone cell effect \vkh minimal effects on blood pressure and smooth mus-
cle, including cardiac muscle. It has further been found that this effect can be obtained by using a synthetic PTH containing only
the first 34 amino acids of PTH. with substitution at the twenty-fifth, twenty-sixth and twenty- seventh, amino acids as described.
These analogues of PTH also are effective in the treatment of osteoporosis and other bone diseases.

FOR THE PL RFOSES Of ISFORMATiOS OSLY

Coiles u^l 10 iilcntU> Simo parts lo ihe PCI on the front pa^e^ or pamphlets publishing imernaiional
applications under the PCn*.

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FR

f-rancu

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Ntjuri Uinta

AU

UA

MW

Malawi

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t-turcU K:n^hti»^

NL

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BE

BurlLitta ^a'Hl

CN

NtJ

S»ifi*.ay

BK

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NZ

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BC

BttlgpiriJ

MU

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PL

PuUml

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Benin

IE

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PT

Ponu];;tl

BR

Brj/il

IT

lUlv

RO

Romanij

CA

JP

RU

Kuviijn KcilcraluMt

CF

C ciUrjl ArriL.iif Kt:(iiin!.t

KP

I >cKiui.ratt«. Pctijitt'v Rcp .blit

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KR

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CI

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SN

CM

(*a:ttcrtK>n

Lk

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su

Soviet Unurti

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LU

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TO

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CZ

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MC

TC

DC

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MI

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US

UrtitctI Suilir* of America

ES

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Vtti Nam

FI

wo 93/06845

PCr/US92/08477

PARATHYROID HORMONB ANAL0QDE8 BUBBTITPTED AT .
^^2S, 26^ 27 M?D.oeE IN 0BTEOF0RO8IB TREATMENT

FIELD OF THE INVENTION -

This invention relates to analogues of parathyroid hormone
which, by substitution at the twenty-fifth, twenty-six and
twenty-seventh positions of natural parathyroid hormone, have
been found to affect calcium change in bone cells without
producing the typical effects of parathyroid horrtone on
systolic and diastolic blood pressure, the effects on smooth
muscle relaxation, vascular smooth muscle calcium change as
well as positive chronotropic and inotropic effects on the
heart.

BACKGROUND OF THE INVENTION

Parathyroid hormone (hereinafter, PTH) is produced by the
parathyroid gland and is involved in the control of calcium
levels in blood. It is a hypercalceraic hormone, elevating
blood calcium levels. PTH is a polypeptide and the amino. aciS
sequences of bovine and "human PTH are closely related.^ Only
the residues at locations one, seven and sixteen differ between
the two. Synthetic polypeptides containing the first thirty-
four residues of PTH may be prepared using the mjethod disclosed
by Erickson and Merrifield, The Proteins , .Neurath et al,, Eds.,
Academic Press, New York, 1976, page 257, preferably as
modified by the method of Hodges et air, Peptide Research. 1 ,
19 (1988) ,

When serum calcium is reduced to below a "normal" level,
the parathyroid gland releases PTH and resorption of bone
calcium and increased absorption of calcium from the intestine,
as wbII as renal reabsorption of calcium, occyr.

The antagonist of PTH is calcitonin, which acts to reduce
the level of circulating calcium. PTH is known to stimulate
osteoclasts and its activity requires the presence of

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PCT/US92/08477

2

derivatives of vitaroin D3, especially 1,25-
dihydroxycholecaloif erol .

Intracellular calcium, particularly in the ceAs of the
vascular system, has been shown to affect changes in vascular
5 tension, as can be measured by changes in blood pressure. U.S.

Patent Application 603,745 describes one method which has been
discovered to regulate calcium uptake in vascular cells.

osteoporosis is a progressive disease which is
particularly characteristic of postmenopausal women, and
10 results in the reduction of total bone mass. The sequelae

frequently involve fractures of load-bearing bones and the
physical degenerations characteristic of immobilizing injuries,
osteoporosis -is ' associated with hyperthyroidism,
hyperparathyroidism,' Ctishings syndrome and the use of certain
15 steroidal drugs. Remedies historically have involved increase

in dietary calcium, estrogen therapy and increased doses of
vitamin D.

PTH has been used to treat osteoporosis. However, while
tliG use of PTII is effective in the treatment of osteoporosis by

20 diminishing the loss of bone mass, PTH may exliibit other

undesired pharmalogical effects, such as hypotension and smooth
" " muscle relaxation (e.g.. .relaxation of gastrointestinal organs,

uterus, tracheal and vas deferens) as well as positive
chronotropic and inotropic effects on the heart. The

25 relaxation effects of PTH on smooth muscle as well as positive

chronotropic and inotropic effects of PTH are described in Pang
et al. Trends in Pharmacologica l Sciences. Vol. 1 i Ko. 9, pp.
340-341 (September 1986) .

U.S. Patent No. 4,771,124 discloses the property of bovine

30 and human Pl'H analogues wherein Trp^^ is substituted by amino

acids phenylalanine, leucine, norleucine, valine, tyrosine,
beta-naphtylalanine and alpha-naphtylalanine as a PTH
antagonist. While it was suggested that these analogues might
be useful in the treatment of osteoporosis, it was based on the

35 analogues antagonistic action to PTH. Furthermore, there was

no data to indicate the effectiveness these analogues on bone
or other tissue. In addition, analogues with substituted at

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3

Trp^-^ with leucine, phenylalanine or tyrosine would produce
undesired secondary effects of smooth muscle relaxation,
vascular smooth muscle calcium change as well as positive
chronotropic and inotropic effects on the heart.

Because PTH is a peptide, topical administration would be
the preferred method of administration. However, topical
application of PTH or the aforementioned analogues which
exhibit vasoactivity would likely produce an undesired local
vascular reaction. This reaction could be potentially
detrimental if, for example, nasal administration is employed.

It is one object of this invention to ameliorate bone loss
while preventing smooth muscle relaxation as well as positive
chronotropic and inotropic effects on the heart and without
significantly changing blood pressure. It is another object of
this invention to identify that portion of PTH which is
responsible for calcium regulation and that portion which
appears to be primarily related to control of blood pressure
and smooth muscle action.

BRIEF SUMMARY OF THE INVENTION

-Modification of either bovine or human PTH at each of the

twenty-fifth, twenty-sixth and twenty-seventh amino" acid
positions to substitute for -arginine-lysine-lysine- either
alanine, asparagine, aspartic acid, cysteine, glutamine,
glutamic acid, glycine, histidine, isoleucine, leucine,
methionine, phenylalanine, proline, serine, threonine,
tryptophan, tyrosine or valine produces substantially no change
in systolic and diastolic blood pressure, substantially no
change in muscle tension and substantially no change in the
rate of contraction and the force of contraction of the heart
as compared to native PTH. It also has been observed that the
PTH analogue containing only the first thirty-four amino acids,
with substitution at the twenty-fifth, twenty-sixth and twenty-
seventh positions, is equally effective in the "osteo effect"
without changing blood pressure or causing muscle relaxation or
positive chronotropic and inotropic effects on the heart.

wo 93/06845

PCr/US92/08477

The analogues of the present invention should be effective
in ameliorating bone loss while preventing smooth muscle
relaxation as well as positive chronotropic and inotropic
effects on the heart and vfithout significantly changing blood
5 pressure.

BPTKF DES f'PTPTTON OF THR PRAWIKGS
Fig. la shows the structure of natural bovine PTH (SEQ ID

Nb:l).

Fig. lb shows the structure of natural human PTII (SEQ ID
10 NO: 2).

Fig. 2 shows the structure of bPTH (1-34) with each of
positions 25, 26 and 27 substituted with Xaa (SEQ ID NO:3).

Fig. 3 shows the structure of bPTH (1-34) with each of
positions 25 r 26 and. 27 substituted with Ala (SEQ ID NO: 4).
15 Fig. 4 shows the structure of hPTII (1-34) with each of

positions 25, 26 and 27 substituted Xaa (SEQ ID NO:5).

Fig. 5 shows the structure of hPTH (1-34) with each of
positions 25, 26 and 27 substituted Ala (SEQ ID NO: 6).

Fig- 6 shows the structure of bPTII with each of positions
20 25, 26 and 27 substituted with Xaa (SEQ ID N0:7),

Fig. 7 shows the structure o_f bPTIl with each of positions
25, 26 and 27 substituted with Ala (SEQ ID NO: 8) .

Fig. 8 shows tlie structure of hPTH with each of positions
25, 26 and 27 substituted with Xaa (SEQ ID N0:9) .
25 Fig. ,9 shows the structure of hPTH with each of positions

25, 26 and 27 substituted with Ala (SEQ ID NOtlO).

Fig. 10 shows the effect of bPTH- (1-34). and its analogues
on diastolic blood pressure of anesthetized Sprague-Dawley
rats.

30 Fig. 11 shows the effect of bPTH-(l-34) and its analogues

on systolic blood pressure of anesthetized Sprague-Dawley rats.

Fig. 12 shows the vasorelaxing effect of bPTH-(l-34) and
its analogues on rat tail artery helical strip in vitro .

Fig. 13 shows the depolarizing concentrations of KCl which
35 increased calcium ion levels in cultur-ed-osteoblasts . Drug 788

is an anti -osteoporotic agent which inhibits the KCl effect.

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PCT/US9Z/08477

5

Figs. 14 a-d show the depolarizing concentrations of KCl
which increased calcium levels in cultured osteoblasts.
Addition of bPTH-(l-34) inhibits the KGl effect.

Fig. 15 shows the effect of Cs88 [bPTH-(l-34) ] on the mean
arterial blood pressure of anesthetized Sprague-Dawley rats.

Fig. x6 shows the dose-response relationship between Cs 8 8
[bPTH-(l-34) ] and the tension of rat tail artery helical strips
precontracted with KCl, norepinephrine and AVP.

Fig. 17 shows the effect of Cs88 on [Ca^'^jj^ in cultured
UMR osteoblast cells.

Fig. 18 shows the effect o! Cs88 on [Cei^'^]^^ in cultured
UMR cells.

Fig. 19 shows a comparison of the effect of Cs221 and Cs99
and bPTH on the mean arterial blood pressure of anesthetized
Sprague-Dawley rats.

Fig. 20 shows the relation between the relaxation curves
of Sprague-Dawley rat tail artery helical strips, precontracted
with AVP when treated with CslOO, Cs99; Cs88, Csll7 and Cs221.

Fig. 21 shows a comparison between the effect of Cs221 and
hPTH on the intracellular calcium uptake in the presence of KCl
in UMR cells in culture.

Fig. 22 shows the effect of Cs221 on the intracellular
calcium uptake in the presence of KCl in UMR cells in culture.

Fig. 23 shows the effect of CslOOl on the intracellular
calcium uptake in the presence of KCl in UMR cells in culture;.

Fig. 24 shows the effect of Cs221 on the contractility and
contraction rate of right atrial tissue of Sprague^-Dawley rats.

Fig. 25 shows the effect of Cs206l and CslOOl on the
contractility and contraction rate of right atrial tissue of
Sprague-Dawley rats.

DETAILED DESCRIPTION OF THE INVENTION

There are at least two known catagories of functions for
PTII. PTII is involved in calcium balance in the blood stream
and controls both the amount of calcium uptake from the

/06845

PCT/US92/08477

astrointestinal tract and the deposition and removal of
alcium from bone. Calcium also has been found to be effective
n the maintenance of blood pressure. Cox, .T , Cardiovascular
h.rmacoloav . vol. 8 (1986), Supp. 8 S48. Control of calcium xn
le walls of blood vessels is a useful therapeutic regimen for
ontroliing hypertension and calcium channel blockers , which
revent the introduction of calcium into cell walls, is a
^nventional therapy for hypertension. Needleman et al. xn
Dodman and Oilman's The PharmacologiraT Basis of Therapeutics ,
acHillan, New York, (1985), page 816 ff.

Adndnistration of therapeutic doses of PTH has been found
, be effective for the control of osteoporosis, particularly
. individuals Who have been subjected to thyroidectomies/
.rathyroidectomies. Therapeutic dosages of PTH will, in some
.dividuals, result in unacceptable diminution of blood
-essure and may result in relaxation of smooth muscles such as
.strointestinal, uterus, tracheal, vas deferens as well as
<hibit positive chronotropic and inotropic effects on the
,art TO avoid hypotensive effects, smooth muscle relaxation
Jfects and positive chronotropic and inotropic effects on the
.art, it was envisaged that the structure of PTH could be
Edified to decouple the hypotensive, smooth musole relaxation,
nd positive, chronotropic and inotropic function from the bond
alcium and bone deposition function. It has now been
iscovered that a critical site exists at amino acid twenty-
ive twenty-six and twenty-seven, which is -Arg-Lys-Lys- m
oth bovine and human PTII. Substitution at the -Arg-Lys-Lys-
ite with -Ala-Ala-Ala- diminishes the hypotensive as well as
„ooth muscle relaxation and positive chronotropic and
notropic effects without denigrating from the osteo effect,
•hese results suggest that substitution at the -Arg-Lys-Lys-
:ite with amino acids other than basic amino acids arginin% and
ysine would also diminish the hypotensive, smooth muscle
elaxation and positive chronotropic and inotropic effects
dthout denigrating from the osteo effect.

The procedure of Erickson and Merrifield, as modified by
todaes.et_al^, as described above, may be used to synthesize

wo 93/06845 PeT/US92/08477

According to the method, Ca^"*" which is present in the ceil can
be quantified by exciting the dye at two different wavelengths,
•340 and 380 nm. The emission fluorescence is measured at 510
nm. The calcium concentration is proportional to the ratio of
the fluorescent emission when excited at 340 nm to the emission
at 380 nm. It is conventional to report the concentration of
calcium within the cell in terms of the fluorescence ratio, the
340/3.8 0 ratio. This technique is described in Grynkiewicz et
al., J. Biol, Chem. . 260 . 3440 ( 1985) and Pang et al. , P. N, A.
S, fUSAV. 07 , 623 (1990).

Figs. 13, 14 a-d, 17, 18, 21, 22 and 23 illustrate the
results of the above-described, measurements when inhibitors
such as an anti-osteoporotic agent (788) or bPTH-(l-34)' fer
Csll4 were used in the presence of KCl.

As can be readily seen from the figures, the PTH
analogues, whether full length or 1-34, which contain anomalous
amino acids at positions twenty-five, twenty-six and twenty-
seven (most particularly those which contain Ala -Ala -
Ala^*'), do not effect a hypotensive and smooth muscle
relaxation response, including positive chronotropic effects,
but do inhibit calcium uptake as stimulated by KCl in
_ osteoblasts,^ which indicates that these compounds would have
the same effect on bone cells' as PTH and~ would be useful in -the_
treatment of osteoporosis in mammals and, particularly, in man,
without the af ormentioned deleterious side effects in the
elderly.

While not being bound by any theory, it is suggested that
substitution Arg^^-Lys^^-Lys^'^ by other amino acids in 1-84 PTH
and in the 1-34 analogues removes the vasodepressor, - smooth
muscle relaxation and positive . chronotropic and inotropic
effects of either bPTH or hPTH. The effect on KCl induced
calcium uptake in osteoblasts, however, is essentially
unchanged for 1-84 or 1-34 PTH. In other words, the effect on
bone cells is unchanged from PTH.

The physiological significance of an inhibiting effect on
the KCL induced calcium uptake in bone cells is not yet
understood. One hypothesis is that the analogues interact

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10

fully with bone cell receptor activity. The fact that the same
effect is seen for" both PTII and the analogues disclos.ed herein
suggests that the site of interaction with the osteoblast cell
receptor is unchanged by the substitution.
5 The analogues of the present invention can be used in the

treatment of osteoporosis and other bone related diseases and
disorders involving bone cell calcium regulation.

The analogues of the present invention may be administered
to a warm-blooded mammalian in need thereof, particularly a.

10 human, by parental, topical, rectal administration or by

inhalation. The analogues mai be conventional!, fcrrpulated xn
a parenteral, dosage form compounding about 1 to about 300 mg
per unit of dosage with a conventional vehicle, excipienn,
binder, preservative, stabilizer, color, agent or the like as

15 called for by accepted pharmaceutical practice.

For parental administration, a 1 to 10 ml intravenous,
intramuscular or subcutaneous injection would be given one to
four times daily. The injection would contain an analogue of
the present invention in an aqueous isotonic sterile soliitxon

20 or suspension optionally with a preservative such as phenol or

a solubilizing agent such as ethylenediaminetetraacetxc acid
- - - - (EDTA) -. Among -the _ acceptable vehicles and solvents that may be
employed are water. Ringer's solution and isotonic- . sodium,
chloride solution. In addition, sterile, fixed oils are

25 conventionally employed as a solvent or suspending medium.

synthetic monoglycerides , diglycerides , fatty acids (such as
oleic acid) find use as fixed oil in the preparation of
injectables.

For rectal administration, the analogues of the present
30 invention can be prepared in the form of suppositories by

mixing with a suitable non-irritating excipient such as cocoa
butter or polyethylene glycols.

For topical use, the analogues of the present invention
can be prepared in the form of ointments, jellies, solutions,
35 suspensions or dermal adhesive patches.

in a powdered aerosol, analogues of the present invention
may be administered by a spinhaler turbo-inhaler device

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11

obtained from Fisons Corporation of Bedford, Massachusetts, at
a rate of about 0.1 to 50 mg per capsule, 1 to 8 capsules being
administered daily for an average huinan. In a liquid aerosol,
the compounds of the present invention are administered a,t the
rate of about 100 to 1000 micrograms per "puff" or activated
release of a standard volume of propellSint. The liquid aerosol
would be given at the rate of 1 to 8 "puffs" per day with
variation in dosages due to the severity of the . conditions
being treated, the weight of the patient and the particle size
distribution of the aerosol. A fluorinated hydrocarbon or
isobutane find use as propellents for liquid aerosols.

Daily, doses are in the range of about 0.01 to about 200 mg
per kg of body weight, depending on the. activity of- the
specific compound, the age, weight, sex and conditions of the
subject to be treated, the type and severity of the disease,
the frequency and route of administration. As would be well
known, the amount of active ingredient that may be combined
with the carried materials to produce a single dosage will vary
depending : upon the host treated and the particular , mode of
administration.

The following examples demonstrate the utility of
- applicants' invention. The examples are not limiting, but are
. illustrative only, and modifications whibh would be apparent ta
those skilled in the art are included within the scope of this
disclosure.

Example 1

In Vivo Blood Pressure Measurement.

Sprague-Dawley (S-D) rats were anaesthetized with
pentobarbital and a cannula was inserted into the carotid
artery. The rats were kept sedated during tte pfbcedux^e and
were injected with PTH peptides only when the blood pressure of
the rats were stable. Peptides were injected through a cannula
in the jugular vein, in amounts of 1, 3 and 5 or more /ig/kg and
the mean systolic and diastolic blood pressure was monitored
continuously throughout the procedure. Results are reported
with comparison to bPTH-(l-34) ,

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PCr/US92/08477

Byamole 2

Tn Vitro Rat Tail Artery Helical Strip Tension Assay

The assay was performed according to Pang et al., SleM
vo.^P.ls. 22, 57 (1985) . Spragae-Dawley rats were anaesthetized
5 with pentobarbital and the tail artery excised and placed in

ice-cold Krebs-Hanseleit solution (KHS) oxygenated with 95%
5^ CO,. Each artery was. cut helically and strips of
approximately 1.5 cm were secured in a, sawyer-Bartlestone
^ chana,er containing KHS. The force generated by the strips was
10 measured with a Grass FT03 force displacement transducer and

recorded on a polygraph. Isolated tail artery helical strips
were equilibrated for 1 hour prior. to use.

one to two minutes prior to addition of a peptide,- thB
strips were contracted by addition of either arginine
15 vasopressin (AVP) , potassium chloride (KCl) or norepinephrine

(NE) to the bath. The peptide was then added to the bath and
the degree of relaxation measured. Bovine serum albumin was
used as a control. Results are reported as percent decrease m
tension for each drug and dose used. Drug dose is calculated
20 on the basis of the final concentration in the bath solution..

FVaTm ple 3 - ^ . _ „ _

Tn Vitro atrial contractility and contraction rate measurement. -
The assay was performed according to Tenner et al.,
,,...^4.n .Tourn.-' PhYc-^moav Rnd Pharmacology, Vol. 61, No.

25 10 (1983) pp. 1162-1167. Sprague-Dawley rats weighing between

100 and 250 g were treated with heparin (500 lU, i.p.) 15
minutes prior to decapitation. Thoracotomies were performed
and the heart rapidly excised and placed in a cold
physiological salt solution (PSS) having the following

30 composition (in millimolar) : HaCl, 120; KCl, 5.63; CaCl2, 2.0;

MgCl2, 2.1; NaHCOj, 25.0; dextrose, 9.7. The solution was
continously aerated by a gas mixture of 95% 02'5i CO^. The
right atrium was isolated and suspended in a tissue chamber
containing 20 mL of PSS at 37.C, pH 7.4. iVtria were allowed to

35 equilibrate for 1 hr under a resting t nsion of 1 g.

The atrial rate and force were determined from

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13

contractions recorded by a Grass FT. 03 force-displacement
transducer and a Grass model 79 polygraph*. The Basial atrial
rate for control atria (as determined by counting the frequency
of contractions) was 258 ± 7 bpm {n=29) . Basal developed force
of the spontaneously beating right atria was 0.33 ± O.QS g
(h=10) . Dose-response curves for the peptides were obtained by
cumulative addition of the respective peptides. Drug dose is
-calculated on the basis of the final concentration in the bath
solution.

**

Example 4

Measurement of Intracellular. Free Calcium Concentration In
vitro

Intracellular free calcium concentration was measured
using the fluorescent dye FURA-2 according to the method of
Grynkiewicz at al., J. Biol. Chem. , 260 , 3440 (1985) and Pang
et al.,. P. N. A. S, fUSA) , 87 . 623 (1990) . UMR-IQS rat:
osteosarcoma ceils (ATCC CRL-1661) are incubated in 1-10 fiVT
FURA-2 AM (Sigma Chemical Co., St. Louis), the acetoraethoxy
ester of FUHA-2. Upon hydrolysis within the cell, FURA-2 is
released which selectively binds to free Ca^**". Binding to-Ca^"^
shifts the fluorescent spectrum of FURA-2. Quantitation is
obtained by exciting the dye at two different wavelengths;
preferably 34 0 and 380 nm and measuring the fluorescent
emission at 510 nm. The concentration of calcium is
proportional to the ratio of the fluorescence emitted at 340 nm
to that at 380 nm.

KCl is used in the medium to stimulate [Ca^***]j^ increase.

After the intracellular [Ca^'^]^ had been, measured^ the
cells were washed with the original medium and the analogues
added and the intracellular [Ca^*^]j^ measured again. KCl was
then added without washing to measure the effect of the
analogue on KCl induced [Ca^^Jj^ changes. After measurement,
the cells were washed with the medium 3-4 tiiries and KCl again
added to determine the recovery of the cells after removal of
the analogue. Results are shown by actual ttaces and
histograms summarizing the results. As can be seen from Figs.

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14

14 a-d, PTH inhibits intracellar [Ca^+jj^ increases as
stimulated by KCl and measured by the method. Pigs. 18, 21, 22
and 23 illustrate comparable results for the aa25/26,27
analogues.

The comparability of the analogues and PTii itself is
considered to indicate that the analogues would be as useful as
PTH for the treatment of osteoporosis.

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PCr/US92/08477

Designation

CS8S
Cs99
CslOO
4:sii7

Cs 221
CsXOOl

cs2oai

15
Table I

Length

1-34
1-34
1-34
1-34
1-34
1-34
1-84

Source

bovine

bovine

bovine

bovine

human

human

human

Substitution Site

none
Ala
Ala
Ala

25
26
27

Ala 25,26,27

none

none

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16

PCr/US92/08477

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i> APPLICANT: PANG, Peter K.T.

JIE, Shan

fii) TITLE OF INVENTION: PARATHYROID HORMONE ANALOGUES
SUBSTITUTED AT AA ^' ^' AND USE IN OSTEOPOROSIS
TREATMENT

(iii) \NDMBER OF SEQUENCES: 10

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Nikaido, Marmelstein, Murray & Oram

(B) STREET: 655 Fifteenth Street N*W. , Suite 330

(C) CITY: Washington D,c.

(D) STATE:

(E) COUNTRY: United States of America

(F) ZIP: 20005-5701

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE : Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/HS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version Si. 25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION KUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(Vii) PRIOR APPLICATION DATA: '

(A) APPLICATION NUMBER: US 773,098

(B) FILING DATE: lO-Oct-91

(C) CLASSIFICATION:

(Viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Murray, Robert B.

(B) REGISTRATION NUMBER: 22,890

(C) REFERENCE/DOCKET NUMBER: 1610-2002

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (202) 638-5OO0

(B) TELEFAX: (202) 638-4810

(2) INFORMATION FOR SEQ ID N0:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 84 amino acids

(B) TYPj:: amino acid

wo 93/06845 PCr/US92/08477

17

(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NOrl:

Ala Val Ser Glu lie Gin Phe Met His Asn Leu Gly Lys His
1 5 10

Leu Ser Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu
' 15 . 20 25

Gin Asp Val His Asn Phe Val Ala Leu Gly A1& Ser lie Ala

30 35 40

Tyr Arg Asp Gly Ser Ser Gin Arg Pro Arg Lys Lys Glu Asp

45 • 50 55

Asn Val Leu Val Glu Ser His Gin Lys Ser Leu Gly Glu Ala

60 65 70

Asp Lys Ala Asp Val Asp Val Leu' lie Lys Ala Lys Pro Gin

75 80

(2) INFORMATION. FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 84 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) .SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Ser Val Ser Glu lie Gin Leu Met His Asn Leu Gly Lys His
15 10
Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu
15 20 25

WO93/06845 PCr/US92/08477

18

Gin Asp Val His Asn Phe Val Ala Leu Gly Ala Ser lie Ala

30 35 *° i

Tyr Arg Asp Gly Ser Ser Gin Arg Pro Arg Lys Lys Glu Asp

45 50 55

Asn Val Leu Val Glu Ser His Gin Lys Ser Leu Gly Glu Ala
60 65 70

Asp Lys Ala Asp Val Asp Val Leu He Lys Ala Lys Pro Gin

75 80

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECai£ TYPE: peptide

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Ala Val Ser Glu He Gin Phe Met His Asn Leu Gly Lys His
1 5 . 10

Leu Ser Ser Met Glu Arg Val Glu Trp Leu Xaa Xaa Xaa Leu

15 20 25

Gin Asp Val His Asn Phe
30

(2) INFORiaTION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTHr 34 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

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19

PCr/US92/08477

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Ala Val Ser Glu lie Gin Phe Met His Asn Leu Gly Lys His
1 5 10 .

Leu Ser Ser Met Glu Arg Val Glu Trp Leu Ala Ala Ala Leu
15 . 20 25

Gin Asp Val His Asn Phe
30

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) ' LENGTH: 34 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) HOIJICULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ IDN0:5:

■ Ser Val Ser Glu lie Gin Leu Met His Asn Leu Gly Lys His

1 . 5 10

Leu Asn Ser Met Glu Arg Val Glu Trp Leu Xaa Xaa Xaa Leu
-15 20 25

Gin Asp Val His Asn Phe
30

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Ser Val Ser Glu lie Gin Leu Met His ASn Leu Gly Lys His

1 5 10

wo 93/06845 PCr/US92/ 08477

20

Leu Asn Ser Met Glu Arg Val Glu Trp Leu Ala Ala Ala Leu
15 20 25

Gin Asp Val His Asn Phe
30

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 84 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(Xi) SEQUENCiE DEisCRIPTION: SEQ ID NO: 7:
Ala Val Ser Glu He Gin Phe Met His Asn Leu Gly Lys His

1 5 - ^°

Leu Ser ser Met Glu Arg Val Glu Trp Leu Xaa Xaa Xaa Leu .
15 20 25

Gin Asp Val His Asn Phe Val Ala Leu Gly Ala Ser He Ala

30 35 40

Tyr Arg Asp Gly ser ser Gin Arg Pro Arg Lys Lys Glu Asp

45 50 55

Asn Val Leu Val Glu Ser His Gin Lys Ser Leu Gly Glu Ala
60 65 70

Asp Lys Ala Asp Val Asp Val Leu lie Lys Ala Lys Pro Gin

75 80

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 84 amino acids

(B) TYPE: amino acid

SUBSTITUTE SHEET

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21

(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Ala Val Ser Glu He Gin Phe Met His Asn Leu Gly Lys His
15 10

Leu Ser Ser Met Glu Arg Val Glu Trp Leu Ala Ala Ala Leu

^15 20 - -25

Gin Asp yal His Asn Phe Val Ala Leu Gly Ala . Ser He Ala

f

30 35 - .40

Tyr Arg Asp Gly Ser Ser Gin Arg Pro Arg Lys Lys Glu Asp

45 50 55

Asn Val Leu Val Glu Ser His Gin Lys Ser Leu Gly Glu Ala

60 65 70

Asp Lys Ala Asp Val Asp Val Leu He Lys Ala Lys Pro Gin

75 80

(2) INFORMATION FOR SEQ ID NO: 9:

(i). SEQUENCE CHARACTERISTICS:

(A) LENGTH: 84 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Ser Val Ser Glu He Gin Leu Met His Asn Leu Gly Lys His
1 5 10

Leu Asn Ser Met Glu Arg Val Glu Trp Leu Xaa Xaa Xaa Leu
15 20 25

PCTAJS92/08477

WO 93/06845 21a

Gxn Asp Val HIS Asn Phe Val Ala Leu Gly Ala Ser He Ala

30 35 40

Tyr Arg Asp Gly Ser Ser Gin Arg Pro Arg Lys Lys Glu Asp

" 45 50 55

Asn Val Leu Val Glu Ser His Gin Lys Ser Leu Gly Glu Ala
60 65 70

Asp Lys Ala Asp. Val Asp Val Lea He Lys Ala' Lys Pro Gin

75 80 .

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 84 amino ,acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Ser Val Ser Glu He Gin Leu Met His Asn Leu Gly Lys His

1 5 10

Leu Asn Ser Met Glu Arg Val Glu Trp Leu Ala Ala Ala Leu
15 20 25

Gin Asp Val His Asn Phe Val Ala" Leu Gly Ala Ser He- Ala..
30 35 40

Tyr Arg Asp Gly Ser Ser Gin Arg Pro Arg Lys Lys Glu Asp
45 50 55

Asn Val Leu Val Glu Ser His Gin Lys Ser Leu Gly Glu Ala
60 65 70

Asp Lys Ala Asp Val Asp Val Leu He Lys Ala Lys Pro Gin

75 80

wo 93/06845

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22
CLAIMS

!• A^bovine parathyroid hormone analogue comprising the
structure shown in SEQ ID NO: 3, wherein each of Xaa^^, Xaa^^
and Xaa^^ is Alanine (Ala), Asparagine (Asn) , Aspartic acid
(Asp) , Cysteine (Cys) ^ Glutamine (Gin) / Glutamic acid (Glu) ^
Glyc^e (Gly) , Histidine (His) , Isoleucine (lie) , Leucine
(Leu) , Methionine (Met)> Phenyalanine (Phe) , Proline (Pro) ,
Serine (Ser) , Threonine (Thr) , Tryptophan (Trp) , Tyrosine (Tyr)
or Valine (Val) .

2> -The bovine parathyroid hormone analogue haying the
structure ftliown in SEQ ID NOM. • . . ' .

■ ■

3. A human parathyroid hormone analogue comprising the
structure shown in SEQ ID NO: 5, wherein each of Xaa^^, Xaa^^
and Xaa^"^ is Alanine (Ala) , Asparagine (Asn) , Aspartic acid
(Asp) f Cysteine (Cys) , Glutamine (Gin) , Glutamic acid (Glu) ,
Glycine (Gly), Histidine (His), Isoleucine (lie), Leucine
(Leu), Methionine (Met), Plienyalanine (Phe), Proline (Pro),
Serine (Ser) Threonine (Thr), Tryptophan (Trp), Tyrosine (Tyr)
or Valine (Val). •

4. The human parathyroid hormone analogue having the
structure shown in SEQ ID NO: 6.

5. A bovine parathyroid hormone analogue comprising the
structure shown in SEQ ID NO: 7, wherein each of Xaa^^, Xaa^^
and Xaa^^ is Alanine (Ala) , Asparagine (Asn) , Aspar^tic acid
(Asp), Cysteine (Cys), Glutamine (Gin), Glutamic acid (Gluj ,
Glycine (Gly), Histidine (His), Isoleucine (IJ.e) , Leucine
(Leu) , Methionine (Met) , Phenyalanine (Phe) , Proline (Pro) ,
Serine (Ser) , Threonine (Thr) , Tryptophan (Trp) , Tyrosine (Tyr)
or Valine (Val) .

6. The bovine parathyroid hormone analogue having the
structure shown in SEQ ID NO: 8,

WO93/06843 PCT/IIS92/08477

23

7. A human parathyroid hormone analogue comprising the
structure shown in SEQ ID NO: 9, wherein each of Xaa^^^ ^aa
and Xaa27 Alanine (Ala), Asparagine (Asn) , Aspartic acid
(Asp), cysteine (Cys) , Glutamine (Gin) , Glutamic acid (Glu) ,
Glycine (Gly) . Histidine (His), Isoleucine (He), Leucine
(Leu), Methionine (Mat), Phenyalanine (Phe), Proline (Pro),
Serine (Ser) , Threonine (Thr) , Tryptophan (Trp) , Tyrosine
(Tyr) or Valine (Val) .

8. The human parathyroid hormone analogue having the
structure shown in SEQ ID NO: 10.

9. A pharmaceutical composition comprising a PTII .
analogue according to any one of claims 1-8 and a
pharmaceutically acceptable carrier.

10. A method of treatment of osteoporosis in a patient
in need of such treatment without causing substantial
induction of hypotension, smodth muscle relaxation and
cardiac inotropic and chronotropic action, said method
comprising administering an osteoporotic effective amount of

- a- PTII analogue according to any one of claims 1-8.

wo 93/06845

1/19

PCT/US92/08477

Fig, la

H^N-Zy^-Val-Ser-Glu-Ile-Gln-Wie-Met-nis-Asn-Leu-Gly^
Lys-Ifis-Leu-Ser-Ser-Met"Glu-Arg-Val-Glu-Trg-LeU"Arg-
Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-Val-AXa-Leu-Gly-
Ala-Ser-Ile-Ala-Tyr-Arg-Asp-Gly-Ser-Ser-Gln-Arg-Pro-
Arg-Lys-Lys-Glu-Asp-Asn-Val-Leu-Val-Glu-Ser-niS"Gln-
Lys-Ser-Leu-Gly-Glu-Ala-Asp-Lys-Ala-Asp-Val-Asp-Val-
Leu-lle-Lys-Ala-Lys-Pro-Gln-CO-H

Fig*

H^N-Ser-Val-Ser-Qlu^Ile-Gln-Leu-MetHtis-^^

Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Tgg-Leu-Arg-

Lys-Lys-Leu-Gln-Asp-Val-nis-Asn-Phe-Val-Aia-Leu-Gly-

Ala-Ser-Ile-Ala-Tyr-Arg-Asp-Gly-Ser-Ser-Gln-Arg-Pro--

Arg-Lys-Lys--Glu-Asp-Asn-Val-Leu-Val-Giu-Ser-nis"Gln-

Lys-Ser-Leu-Gly-Glu-Ala-Asp-Lys-Ala-Asp-Val-Asp-Val-

Leu-Ile-Lys^Ala-Lys-Pro-Gln-CO^H

Fig, 2

HpN-Ma-Val-Ser-Glu-llG-GIn-PlLe-Met-Ilis-Asn-Leu-Gly-
Lys-lMs-i^u-§er-Ger-Met-Glu-Arg-Vnl--GJ u-Trp-Leu-Xaa-
2fla^Ma-Leu-Glir-A&p-Val-IIis-Asn-phe--CO«II

Fig. 3

irpN-iUa-Val-Ser-Glu-Iie--Gln-Piie-Met-]li.s-Asn
Lys-iris-Leu-Ser-Ser-Met'-Glu-Arg-Val-GUi-Trp-L^^
Ala-Ala- LeU"Glii-Asp"Val-His-Asn-Phe-C02n

Fig, 4

H^N-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-llis-^^

Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Xaa-

^il-Mi"^®""Gln-Asp-Val-His-Asn-Phe-CO-;H

Fiq> 5

"^^'-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-llis-Asn-Leu-^

liys-I!is-Leu-Asn-Ser-Met-GlU"Arg-Val-Glu-Trp-Leu-Ala-

MS^MS~Leu-Gln-Asp-Val-His-Asn-Phe-C02H

wo 93/06845

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PCr/US92/08477

Fig. 6

lf;,H-Al^-Val-Ser-Glu-He-Gln-Phe-Met-»is-Asn-Leu-Gly-
Lys-Mis-Leu-Ser-Ser-Met-Glu-Arg-Vnl-Glu-Trp-Leu-Xaa-
Ma^^'l-eu-Gln-Asp-Val-llis-Asn-Phe-ValrAia-Leu-Giy-
Ala-Ser-ile-Ala-Tyr-Arg-Asp-Gly-Ser-Ser-Gln-Arg-j?ro-
Arg-Lys-Lys-Glu-Asp-Ash-Val-Leu-Val-Glu-Ser-His-Gln-
Lys-Ser-Leu-Gly-Glu-Ala-Asp-Lys-Ala-Asp-Val-Asp-Val-
LeurHe-Lys-Ala-Lys-Pro-Gln-CO,H

rig. 7

ripN-Ala-Val-Ser-Glu-Ile-Glii-Phe-Met-nis-Asn-Leu-Gly-
Lys-Ifis-Leu-Ser-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Ala-
M5"Ma~Leu-Gln-Asp-Val-nls-Asn-Plie-Val-Ala-Leu-Gly-
Ala-Ser-Iie-Ala-Tyr-Arg-Asp-Gly-Ser-Ser-Gln-Arg-Pro-
Arg-Lys-Lys-Glu-Asp-Asn-Val-Leu-Val-Glu-Ser-Ilis-Gln-
Lys-Ser-Leu-Gly-Glu-Ala-A;p-Lys-Ala-Asp-Vai Asp-Val-
Leu-Ile-Lys-Ala-Lys-Pro-Gln-CO,H

Fig, 8

IIpN"Ser-Val-Ser"Glu-Ile-Gln-Leu-Met"nis-Asn--Leu--Gly- -

Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Xaa- '

^JS~2M'"LeU"Gln"Asp-Val-His-Asn-Phe-VaI--Ala-Leu-Giy-

Aia-Ser-Ile-Ala-Tyr-Arg-Asp-Gly-Ser-Ser-Gln-Arg-Pro-

Arg-Lys-Lys-Glu-Asp-Asn-Val-Leu-Val-Glu-Ser-iiis-Gln-

Lys-Ser-Leu-Gly-Glu-Ala-Asp-Lys-Ala-Asp-Val-Asp-Val-

Leu-IIe-Lys-Ala-Lys-Fro-Gln-CO^jr

rig. 9

H^N-Ser-Va-l-Ser-Glu-IlG-Gln-Leu

• r.ys"llis-Leu-Asn-Ser-Met"Glu-Arg-Val-GlU"Trp-LeU"M
to-to'-^^^-Gi'^-^^^^P-Val-Hls-Asii-Phe-Val-Aln-Leu-Giy- !
Aia-iSer-Ile-Ala-Tyr-Arg-Asp-Gly-Ser-Ser-Gln-Arg-Pro"
Arg-Lys-Lys-Glu-Asp-Asn-Val-Leu-Val-Glu-Ser-IIis-Gln-

Lys-Ser-Leu-Gly-Glu-Ala-Asp"Lys-Ala-Asp-Val-Asp-Val"
Leu-lle-Lys-Ala-Lys-Pro-Gln-CO^II

wo 93/06845

PCr/US92/08477

3/19

sr (D cvj io o

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DIASTOLIC BLOOD PRESSURE ( mmHg )

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PCT/US92/08477

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SYSTOLIC BLOOD PRESSURE ( mmHg )

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PCT/US92/08477

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R
A
T
I

0

4.00001

3.2500-

2.5000-

1.7500
1.0000

CONTROL KC L I 5m M

NEW DISH

PTH 2.5XI0-6M

KCL 15m M AFTER PTH lOMIN
KCL l5mM AFTER WASH

100 200

FIG. 14a

300 400
TIME (sec)

500 600

R
A
T
I

0

1 .7500

1.3750-

1 .0000

A CONTROL KCL l5mM

B PTH 3x 10-7 M

C KCL l5mM (AFTER PTH I5MIN)

D KCL l5mM (AFTER PTH 25 MIN )

0 100 200 300 400 500 600

TIME (sec )

FIG. 14c

wo 93/06845

PCr/US92/08477

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6.7670

A CONTROL KCL 15 mM
B PTH 2.5x lO-6M
C KCL I5mM AFTER PTH
lOMIN

D KCL 15m M AFTER WASH

0.0000

100

200 300 400
TIME(sec )

500 600

FIG. 14b

2. 5000 n

R
A
T

2.1250

L7500

L3750

LOOOO.

A CONTROL KCL l5mM NEW

DISH

B PTH 2.5x10-7 M

C KC L 15 m M ( A FTER F*t H
15 WIN )

100

200 300 400
TIME (sec)

500 600

FIG. I4cl

SUBSTITUTE SHEET

wo 93/06845

PCr/US92/08477

SUBSTITUTE SHEET

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